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Cellular and Molecular Bioengineering

Cellular and Molecular Bioengineering - Poster Session C

Poster H16 - Increasing flexibility and availability of Induced Pluripotent Stem Cells- Endothelial Cells protocol

Friday, October 25, 2024

10:00 AM – 11:00 AM EST

Location: Exhibit Hall E, F & G

Presenting Author(s)

Arshitha Rayanki

Undergraduate Student
University of Texas at Austin, United States

Co-Author(s)

Bryce Larsen

Graduate Student
University of Texas at Austin, United States
Sogu Sohn, MS (he/him/his)

PhD Candidate
University of Texas at Austin, United States

Primary Investigator(s)

Janet Zoldan

Associate Professor
The University of Texas at Austin Biomedical Engineering, United States

Introduction: Induced Pluripotent Stem Cells(iPSCs) are integral to regenerative medicine due to their patient specificity and their ability to differentiate into a variety of cell types. These cells can be used to build tissue, but perfusion in these tissue engineered constructs remains a critical challenge. Often, strategies employ a co-culture of both a target cell type, and endothelial progenitor cells (EPs) with the aim of creating vascularized tissue. iPSCs can differentiate into EPs which further differentiate into the two main types of vascular cells, endothelial cells (ECs) and smooth muscle cells (SMCs). Numerous protocols exist for generating iPSC-EPs, but the cells they produce need to be added to co-cultures after iPSC-EP differentiation but before terminal EC or SMC differentiation. This means that for co-culture conditions, researchers must manage multiple cell type differentiations and timelines which can lead to complications and difficulties in aligning availability of the differentiated cells. This creates a barrier of using multiple cell types in tissue engineering and the viability of these cells after a certain period of time. We aimed to investigate an existing iPSC-EP protocol to achieve flexibility of differentiation timing, extending mature cell availability, and preserving the progenitor state in iPSC-EPs.

Materials and

Methods: iPSCs (iPS-DF19-9-11T.H) from Wicell were grown on vitronectin coated plates from ThermoFisher with E8 until confluent. Once confluent, the cells were passaged and plated on Geltrex LDEV-Free, hESC-Qualified, Reduced Growth Factor coated plates at 10,000 cells/cm^2. The cells were fed E8 for one day after passaging. Differentiation was induced by feeding the cells (DMEM/F-12, B-27, N2), with 150 nM BIO, 30ng/ml BMP4, 25 ng/ml Activin A for 2 days to promote mesoderm formation. Then for 3 days the cells were fed with differentiation media with differentiation media,(DMEM/F-12, B-27, N2), with 2 µM SB431542 and 50 ng/ml VEGF for either 2 or 3 days to increase endothelial progenitor yield. iPSC-EPs were then sorted for CD34 expression on either day 4 of the 5 day protocol or day 5 using a FITC-CD34 antibody and FACS. A CD34- population was also collected. After sorting, iPSC-EPs were plated on a geltrex plate for experiments investigating progenitor state preservation, or vitronectin coated plates investigating bipotency of day 4 and 5 iPSC-EPs. For progenitor state preservation, iPSC-EPs were grown in EGM1( Endothelial Cell Growth Media) , EMG1+ 2 units of VEGI, EMG1 + 4 units of VEGI, and EMG1 + 8 units of VEGI. For bipotency experiments, iPSC-EPs were fed with Endothelial Growth Media 2. The CD34- population was plated onto vitronectin coated plates and fed with Smooth Muscle Growth Media.

Results, Conclusions, and Discussions: Through our experiments we found that iPSC-EPs sorted on day 4 and day 5 both exhibited endothelial cell maturation with VE-Cadherin tight junctions, interspaced with SMCs (Fig. 1). However, the relative yield of CD34+ cells sorted on day 4 was 2.7 times lower than the relative yield of CD34+ cells sorted on day 5. These results show that although there is a decrease in yield, iPSC-EPs sorted a day early in the protocol behave similarly in culture to those sorted on day 5. This allows greater flexibility on the time of sorting for endothelial progenitor cells utilizing this differentiation protocol. We plan on characterizing the relative proportions of ECs and SMCs to see if there are any significant differences in the populations between day 4 and day 5. Next, we successfully differentiated the CD34- population into SMCs by culturing the negatively sorted population in a smooth muscle growth medium for 7 days on vitronectin coated plates. After 7 days, 95.94 ± 3.67% of the population expressed calponin, n=6 (Fig. 2) and found that the CD34- SMCs proliferated and expanded on vitronectin coated plates for at least 3 passages. These results show that after SMC differentiation, we are able to maintain a pure SMCs population with the ability to proliferate across multiple passages. For future work, we hope to co-culture these SMCs with iPSC-EPs in a 3-D environment to see if there is any integration with the microvasculature networks created by the iPSC-EPs. We also have maintained a population of iPSC-EPs after sorting in EGM-1 and VEGi, these cells have expanded over 7 days (Fig. 3) without an obvious endothelial cell monolayer formation as seen formed by iPSC-EPs in EGM2 and given VEGF at 50 ng/ml. We plan on culturing them in a 3D environment to see if they will differentiate and form microvasculature. If so this would suggest that we can preserve their progenitor status by culturing in VEGF free media and inhibiting VEGF.

Acknowledgements (Optional):